PTEN is a tumor suppressor that is mutated in many forms of human cancer. PTEN encodes a phosphatase that recognizes the important second messenger, phosphatidylinositol-3,4,5-triphosphate (Pl- 3,4,5-P), and removes the 3'-phosphate from the inositol ring. PTEN therefore antagonizes PI-3 kinase, which phosphorylates the inositol ring at the same position. PI-3 kinase is an oncogene in its own right that is frequently mutated and amplified in tumors. Although PI-3 kinase and PTEN potentially influence many signaling pathways, perhaps the best understood pathway involving these genes is the insulin/IGF pathway, which is conserved in homo sapiens, c. elegans and drosophila. In these species, PTEN acts downstream of the insulin/IGF receptor, the insulin receptor substrate (IRS) adaptor and PI-3 kinase and upstream of AKT kinase. The most significant targets of AKT are the FOXO transcription factor genes. Activation of the PI-3 kinase pathway is highly oncogenic; however, it remains unclear how many components of the pathway contribute to oncogenesis. This application will focus on three fundamental problems in understanding how the PI-3 kinase pathway drives tumor formation. In aim 1, we will explore the possibility that pathological over expression of IRS2 in tumors can stimulate PI-3 kinase and collaborate with other alterations on the Pl- 3 kinase pathway to boost the PI-3 kinase signal. We will introduce IRS2 into normal human cells and examine them for alterations in growth, differentiation, and signaling. The minimal domain that is competent for inducing signaling and altered growth will be defined. We will measure the frequency of over expression in tumors and measure the effect of RNAi on tumor cell line growth and signaling. Lastly, we will establish a transgenic model of IRS2 overexpression to test the hypothesis that it is an oncogene . In aim 2, we will determine the effect of PTEN dose on tumor formation in mice. Loss of one copy of PTEN and reduced PTEN protein expression are extremely common in human malignancy. We therefore will reduce expression of PTEN in mice using RNAi and examine its effect on tumor formation. In aim 3, we will investigate how PTEN regulates FOXO transcription factors to activate a target gene. DNA elements will be identified on the promoter that coordinate gene activation. A combination of overexpression, RNAi, and chromatin immuno- precipitation experiments are planned to identify the mechanism of activation. PERFORMANCE SITE(S) (organization, city, state) Health Sciences Division Columbia University 630 West 168th Street New York, NY 10032 PHS 398 (Rev. 09/04) Page 2 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): ParSOOS, Ramon E KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Parsons, Ramon PARSONSR Columbia University PI Keniry, Megan Columbia University Post-doc Szabolcs, Matthias Columbia University Pathologist OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Hibshoosh, Hanina Columbia University Collaborator Human Embryonic Stem Cells [X] No \_\ Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://StemcellS.nih.gov/reaistrv/index.asp. Use continuation pages as needed. If a specific line cannot be referencedat this time, include a statement that one from the Registry will be used. Cell Line Disclosure Permission Statement Applicable to SBIR/STTR Only. See SBIR/STTR instructions. Yes NO PHS 398 (Rev. 09/04) Page 3 Form Page 2-continued Number the folowing pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Parsons, Ramon E The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description, Performance Sites, Key Personnel, Other Significant Contributors, and Human Embryonic Stem Cells Table of Contents Detailed Budget for Initial Budget Period (or Modular Budget) Budget for Entire Proposed Period of Support (not applicable with Modular Budget) Budgets Pertaining to Consortium/Contractual Arrangements (not applicable with Modular Budget) Biographical Sketch - Principal Investigator/Program Director (Not to exceed four pages) 6-8 Other Biographical Sketches (Not to exceed four pages for each - See instructions) 9-16 Resources 17 Research Plan, 18-42 Introduction to Revised Application (Not to exceed 3 pages) Introduction to Supplemental Application (Not to exceed one page) A. Specific Aims B. Background and Significance 19-21 C. Preliminary Studies/Progress Report/ (Items A-D: not to exceed 25 pages*) 22-30 Phase I Progress Report (SBIR/STTR Phase II ONLY) * SBIR/STTR Phase I: Items A-D limited to 15 pages, D. Research Design and Methods 31-42 E. Human Subjects Research 43 Protection of Human Subjects (Required if Item 4 on the Face Page is marked Yes) 43 Data and Safety Monitoring Plan (Required if Item 4 on the Face Page is marked Yes and a Phase I, II, or III clinical trial is proposed) Inclusion of Women and Minorities (Required if Item 4 on the Face Page is marked Yes and is Clinical Research) Targeted/Planned Enrollment Table (for new and continuing clinical research studies) Inclusion of Children (Required if Item 4 on the Face Page is marked Yes) 43 F. Vertebrate Animals 43-44 G. Literature Cited 44-51 H. Consortium/Contractual Arrangements NA I. Resource Sharing 51 J. Letters of Support (e.g., Consultants) 52-53 Commercialization Plan (SBIR/STTR Phase II and Fast-Track ONLY) Checklist. 54. Appendix (Five collated sets. No page numbering necessary for Appendix.) Check if Appendix is Included Appendices NOT PERMITTED for Phase I SBIR/STTR unless specifically solicited Number of publications and manuscripts accepted for publication (not to exceed 10) Other items (list): PHS 398 (Rev. 09/04) Page 4 Form Page 3